This invention claims priority of the German patent application 100 50 963.0 which is incorporated by reference herein.
The invention concerns a method and an apparatus for optical measurement of a surface profile of a specimen.
Several methods for three-dimensional reconstruction of surfaces of microscopic structures are known. In CLSM (confocal laser scanning microscopy) the specimen is scanned point by point in one plane, and an image with very little depth of focus is thereby acquired. With a plurality of images in different planes, and by way of appropriate image processing, the specimen can be depicted three-dimensionally. In CLSM the data can be generated only in very expensive fashion, and high-quality optical components are a prerequisite. A high level of technical complexity is also necessary in another technique in which a 3D reconstruction is achieved by means of a thin line of light directed onto the surface that is to be reconstructed.
The existing methods are expensive, and require not only good technical understanding but also a great deal of alignment work. In addition, the methods are usable only for small areas.
It is the object of the invention to measure the surface profile of a specimen quickly and economically without contact and without damage to the specimen.
The object is achieved by a method that measures the surface profile of the specimen optically in a three-dimensional coordinate system (x, y, z) and contains the following steps:
acquiring a series of n images of the specimen in different planes in the z direction by means of an image acquisition apparatus, each plane containing a plurality of coordinate points (x, y);
assigning each of the images to a plane in the specimen having a defined plane number;
referring each plane to a common origin (x0, y0) in the coordinate system (x, y, z); and
generating a mask image using the following procedure for each coordinate point (x, y):
comparing the image contents of all n images at that coordinate point (x, y) to one another in order to determine a plane therefrom according to predetermined criteria, assign its plane number to that coordinate point (x, y), and store it in the mask image.
The object is also achieved by way of an apparatus that measures the surface profile of the specimen optically and contains the following features:
an image acquisition apparatus for acquiring a series of n images of the specimen in various planes in the z direction of a coordinate system (x, y, z), each of the images being assigned to a plane in the specimen having a defined plane number, and each plane being referred to a common origin (x0, y0) and each plane containing a plurality of coordinate points (x, y); and
a computer that, for each coordinate point (x, y), compares the image contents of all n images at that coordinate point (x, y) to one another in order to determine a plane therefrom according to predetermined criteria and assign its plane number to that coordinate point (x, y) and store it as a mask image.
With the invention, it is possible to ascertain surface profiles of specimens without a great deal of technical complexity. The image acquisition apparatus can comprise simply a camera. For images of smaller or microscopic specimens, a macroscope or microscope together with a camera placed on it is used as the image acquisition apparatus. The data of the surface profiles that are obtained can easily be displayed on a computer.
In particular, microscopic surfaces can be reconstructed by means of conventional light microscopy. In microscopy, specimens having a geometric surface pattern cannot be imaged simultaneously with complete sharpness. Using a suitable design, different focal planes of the specimen are therefore traveled to in controlled and reversible fashion and images in the various focal planes are recorded, preferably with a digital image. A xe2x80x9ccontour mapxe2x80x9d of the microscopic specimen surface is automatically calculated therefrom using a computer.
Even a simple microscope having an adjustable-height stage and a CCD camera can be sufficient. Images of the specimen are acquired at different stage heights, i.e. in various focal planes. Because of the depth of field of the microscope, each of the images contains sharp and unsharp regions. From the series of images that are acquired, specific criteria are used, for example, to pick out the sharp regions in each of the individual planes and assign them as plane numbers to the corresponding (x, y) coordinate points. The assignment of a plane number to each (x, y) coordinate is stored in a memory as mask image.
The mask image represents a two-dimensional image that contains only essential data. One advantage of the mask image is that the data volume is greatly reduced. On the one hand, because of the reduction in data volume to two dimensions as opposed to the processing of a three-dimensional image, normal and conventional 2D image processing algorithms can be applied to the mask image. On the other hand, the reduced data volume means that the 3D information can be quickly and easily retrieved from the mask image. The plane number at a predefined (x, y) coordinate can be retrieved from the mask image at any time, and a relative elevation value can thereby be displayed. By multiplying the plane number by the distances between the planes, an absolute elevation value can be indicated in a dimensional unit, e.g. in xcexcm. In addition, the relative or absolute elevation values can be assembled into a completely sharp three-dimensional image of the specimen surface. A three-dimensional image reconstructed in this fashion thus exhibits greatly improved image quality as compared to conventional microscopic images. In addition, because of the greatly reduced data volume, the surface profile of the specimen can be read out of the mask image quickly and thus displayed on a monitor in real time. The method is also robust, i.e. it is dependable and has little susceptibility to failure.
Furthermore, the procedure of the calculation of the mask image can be accelerated if a reduced image is used, i.e. only every second or third or forth etc. of the pixels in x- and y-direction is taken into account instead of each pixel. The generation of the mask image can be applied to the reduced image. If necessary, a fast running interpolation algorithm can be applied afterwards.
It is moreover advantageous that any standard light microscope can be used for the method according to the present invention, thus yielding an economical alternative to CLSM.